The replicative history and replicative potential of human naive and memory T cells, critical parameters of lymphocyte biology, were analyzed. Telomeres are unique terminal chromosomal structures which shorten with cell division in vitro and with increased age in vivo for human somatic cells. We found that telomeres were longer in naive T cells than in memory cells from the same donors over a wide range of donor age. This suggests that the differentiation of memory cells from naive precursors occurs with substantial clonal expansion. The in vitro replicative capacity of naive cells was greater than that of memory cells from the same donors. Human CD4+ naive and memory cells thus differ in in vivo replicative history as reflected in telomeric length as well as in their residual replicative capacity. Analysis of telomere length regulation in human B cells demonstrated that germinal center(GC)B cells have significantly longer telomeres than the naive B cells that are their precursors or the memory B cells that are their progeny. These results suggest the novel possibility that normal somatic cells of the B lymphocyte lineage express a mechanism capable of extending telomere length. Such a mechanism might function to extend the capacity for clonal expansion of memory and effector B cells. Telomerase, a ribonucleoprotein enzyme that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells and is absent in most normal human somatic cells. The selective expression of telomerase has thus been proposed to be a basis for the immortality of the germline and of malignant cells. When telomerase activity was analyzed in normal human T lymphocytes, it was found that telomerase is expressed at a high level in thymocytes, at an intermediate level in tonsil T cells, and at a low to undetectable level in peripheral blood T cells. Moreover, telomerase activity was highly inducible in peripheral T lymphocytes by activation through CD3 and CD28. Telomerase may thus play a role in T cell development and in the capacity of lymphoid cells for clonal expansion. In human tonsil B cells, telomerase is expressed at a high level in GC B cells and may provide a mechanism for the telomere lengthening that occurs in differentiation from precursor to GC B cells. Expression of the two genes encoding the necessary and sufficient components of telomerase, RNA template (TR) and reverse transcriptase catalytic component (TERT), has been analyzed, and both mRNAs have been found to be regulated during lymphocyte development and activation. It has been further demonstrated that telomerase enzymatic activity in vivo is determined by factors in addition to steady state RNA levels of TR and TERT. It has been demonstrated that events including phosphorylation of hTERT and translocation of TERT from cytoplasm to nucleus occur concurrent with the induction of telomerase activity in activated T cells. To further study the regulation of telomerase at a transcriptional level, a series of genetically engineered mice have been constructed including: mTERT cDNA transgenics, GFP knock-in as a reporter for mTERT transcriptional activity (also resulting in mTERT knock-out inactivation), and an hTERT BAC transgenic that allows study of human TERT regulation in human versus mouse cellular environments. In addition, the role of telomere-associated proteins TIN-2 and tankyrase-2 is being analyzed through construction of both constitutive and conditional knockouts for each of the corresponding mouse genes. Initial studies indicate that constitutive inactivation of either gene results in early embryonic lethality. Conditional knockouts will be used to analyze the mechanism of these effects. A model system has been established for analysis of the genetic regulation of telomere length in mice. Inter-fertile species of mice were identified which differ significantly in telomere length. Crosses between these species have in initial experiments demonstrated that 1) a mechanism exists for substantial telomere lengthening in somatic cells in vivo, and 2) that species-specific telomere length is regulated by segregating genes that are polymorphic between these species. A locus that has predominant effect on telomere length determination has been mapped to a 5cm region of distal chromosome 2. Studies employing mice genetically deficient in telomerse RNA template have indicated that telomere elongation obsered in vivo is telomerae (TR) dependent and, unexpectedly, have shown that mice heterozygous for TR deficiency have impaired capacity for telomere elongation. Parallel studies using mTERT deficient mice demonstrated that heploinsufficiency is not seen in heterozygotes for this gene, suggesting that TR expression, not TERT expression may be functionally limiting during development in vivo. Telomerase activity has also been analyzed in mice, allowing for the first time an analysis of telomerase activity in T cells undergoing an antigen-specific response in vivo. Substantial upregulation of telomerase occurs transiently following antigen challenge, including challenge with infectious virus, demonstrating that this activity is regulated under physiologic conditions of T cell response. Studies of LCMV viral infection have demonstrated that telomerase activity is induced in responses to primary viral infection and is maintained in memory CD8 T cells as well. Telomere length is maintained without loss in the face of extensive clonal expansion, suggesting that telomerase may compensate for telomere loss under these conditions. Experiments are in progress studying the responses of telomerase-deficient mice to directly assess the role of telomerase in anti-viral immune responses. The role played by telomerase in the process of malignant transformation and cancer cell growth is an area of interest, with relevance both for understanding underlying molecular processes and for design of potential interventions. We have generated transgenic mice that over-express telomerase mTERT and have initiated studies of cancer susceptibility in these mice. Preliminary evidence indicates a dramatic increase in the incidence of breast cancer in a transgenic model of mammary carcinogenesis. The genetic and cytogenetic events underlying this effect are being studied. The function of the ataxia telangiectasia mutated (ATM) gene product in tumorigenesis was studied in a mouse model system of ATM-deficient mice. These mice develop a high incidence of thymic lymphomas with characteristic chromosomal translocations within the T cell receptor (TCR) locus. Studies of mice deficient in recombinase associated gene (RAG) activity as well at ATM demonstrated that susceptibility to tumorigenesis in ATM-deficient mice is generalized, and is not limited to RAG-dependent rearrangements in the TCR locus. The mechanism of this tumorigenesis and the identification of genes involved in translocation are under study. In collaboration with the lab of Thomas Ried (NCI), in situ hybridization with BAC contigs has identified the regions of recombination reproducibly occurring in ATM-deficient mice, and sequencing strategies will next be applied to defining the recombination and the involved genes. These studies may lead to identification of novel and previously undescribed oncogenes.